Chapter 3. Poly (ethylene glycol)-graft-trimethyl chitosan block
3.3 Water solubility of copolymers
(A)
(B)
Figure 3. TGA thermograms of graft copolymers based on mPEG 5 kDa and chitosan/TMC (A) PEG(5k)-g-TMC(400) copolymers.
(a) TMC 400 kDa, (b) PEG 5 kDa, (c) PEG(5k)298-g-TMC(400), (d) PEG(5k)641-g-TMC(400), (e) PEG(5k)679-g-TMC(400).
(B) PEG(5k)-g-chitosan copolymers.
(a) Chitosan 400 kDa, (b) PEG 5 kDa, (c) PEG(5k)243-g-chitosan(400), (d) PEG(5k)386-g-chitosan(400), (e) PEG(5k)515-g-chitosan(400).
graft density was as low as 10% and the concentration was as high as 50 mg/ml.
The PEG moieties presented on the TMC chain increased the hydrophilicity of the copolymer, thus leading to improved solubility. In contrast, no clear solutions were observed with PEG grafted chitosan copolymers at pH 7, despite of high graft densities. This is due to the strong interaction between chitosan and PEG [5,6], as demonstrated by the significantly decreased Tm value in DSC experiments.
3.4 Biocompatibility and properties of the complexes
In addition to the solubility, biocompatibility is of particular importance when a polymer is to be used as a drug carrier. Generally, polycationic polymers are related to cytotoxicity as a consequence of their interaction with the negatively charged components on the cell membrane. Therefore, the cytotoxicity of these new synthesized copolymers was investigated, and the concentration of the copolymers resulting in 50% inhibition of cell growth, i.e.
IC50 value, was calculated. Along with the increase of PEG 5 kDa substitution, the free amino groups on TMC 400 kDa chains decreased, which, together with the steric effect of PEG 5 kDa, led to considerably decreased cytotoxicity, as illustrated in Table 3.
Moreover, complex formation leads to further increase of IC50. This can probably be attributed to the electrostatic interaction between the copolymers and insulin, which decreased the charge density of the copolymers, as demonstrated by the decreasedξ–potentials (Table 3). Another possibility is that complex formation embeds part of the free amino groups, preventing them from direct contact with the cells. It was noticed that with PEG 5 kDa coupling, cytotoxicity of TMC 400 kDa could be decreased more than 30 fold.
PEG(20k)-g-TMC(400) copolymers were prepared in the same manner to investigate the influence of PEG MW on the properties of the copolymer and corresponding insulin complexes. Despite the low substitution ratio,
cytotoxicity of the copolymer was decreased remarkably after PEG 20 kDa coupling, and further after complexation, which can probably be explained by the steric effect of the PEG 20 kDa molecules leading to subdued interaction with cell membrane. However, theξ- potential of PEG (20k)10-g-TMC(400) insulin complex decreased only marginally compared to that of TMC 400 kDa in 6 mM Tris buffer, 18.7±1.7 versus 20.9±1.4. A similar phenomenon was observed with PEI(25k)-g-PEG(20k)1 compared to PEI 25 kDa [25].
Table 3. Properties of the PEG(5k)-g-TMC(400) copolymers and corresponding insulin complexes
IC50(µg/ml) (24 h) Complex properties Polymer Substitution b
(%) Graft ratioc
(%) Pure
polymer Complexes
Mass
ratiod Particle size
(nm) Zeta-potential
(mV) Association efficiency (%) TMC 400 kDa - - 15 50 0.3:1 256.3±0.8 20.9±1.4 78.6±4.9 PEG(5k)298-g-TMC(400) 38.9 75.4 40 100 0.7:1 171.7±1.9 11.2±0.9 81.3±1.1 PEG(5k)640-g-TMC(400) 83.5 88.9 >500a > 500a 1:1 181.8±2.1 2.3±0.3 87.0±6.3 PEG(5k)680-g-TMC(400) 88.7 89.5 >500a > 500a 1.5:1 245.6±2.9 1.7±1.0 96.2±2.1 PEG(20k)10-g-TMC(400) 1.3 33.3 100 500 0.7:1 220.8±4.7 18.7±1.7 71.9 ±2.9 PEG(20k)20-g-TMC(400) 2.6 50.0 >500a > 500a 1:1 256.1±7.0 18.8±0.3 79.3 ±1.7
a The highest concentration investigated.
b Calculation based on the primary amino group content in TMC 400 kDa, 30.83%.
c Calculated as a weight ratio of PEG in the graft copolymer.
d Optimized polymer/insulin mass ratio for complex preparation.
PEG 5 kDa and PEG 20 kDa grafted TMC copolymer insulin complexes were also investigated to gain insight into the effect of copolymer composition.
Based on the preliminary experimental procedure, soluble insulin complexes were prepared at optimal polymer/insulin mass ratios, as listed in Table 3.
Particle size, ξ–potentials and the association efficiency (AE) of various
insulin complexes were also investigated. The particle size was in the range of 150-300 nm, and increased with graft ratio. No significant difference in particle size was observed for the two PEG(20k)-g-TMC(400) copolymer insulin complexes. All of the complexes investigated here were positively charged and ξ–potentials decreased with increasing graft density. This is consistent with the cytotoxicity results.
There have been reports that insulin binds to polymers via its negative charge, i.e. adhesion being facilitated by the interaction between the positively charged polymers and the negatively charged insulin. Based on this hypothesis, a decreased AE of PEGylated copolymers would be expected compared to non-modified polymers. Surprisingly, AE values increased with PEG 5 kDa graft ratio and extremely high association efficiency, that is, 96.22%, was achieved with PEG(5k)679-g-TMC(400) copolymer. A similar result was found in PEG(2k)-g-chitosan DNA nanoparticles [26], and preferential partition of insulin into the PEG phase in PEG-dextran systems was also reported [27].
Previously, Prestwich et al. showed that insulin has a high affinity for PEG rich environement [28]. Furthermore, PEG chains have been shown to complex with protein and stabilize them [29,30]. Therefore it is reasonable to assume that a certain proportion of insulin was retained in the PEG moieties conjugated in the TMC matrix, resulting in a higher AE.
Taking all the above results into consideration, PEGylation with PEG 5 kDa is sufficient to increase the biocompatibility of TMC and the corresponding copolymers are promising insulin carriers.
4. Conclusions
PEGylated trimethyl chitosan 400 kDa copolymers with varying PEG molecular weight and graft ratios were successfully synthesized and demonstrated by FT-IR, 1H NMR, 13C NMR and GPC measurements. Decreased melting temperature of PEG in the copolymers indicated that the two blocks are
miscible and compatible. PEG-g-TMC(400) copolymers were completely water-soluble over the entire pH range, irrespective of PEG MW. Based on cytotoxicity results, the potential of PEG(5k)-g-TMC(400) and PEG(20k)-g-TMC(400) copolymers as insulin carriers was studied. The particle sizes were less than 300 nm andξ-potential decreased with increasing graft density, which was consistent with the decreased toxicity. Insulin association efficiency was PEG graft density dependent, and the value could be as high as 96.22%. Based on the good solubility of the copolymers and the speciality of polyethylene glycol, we believe these copolymers could be used to enhance the therapeutic and biotechnological potentials of other macromolecules.
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